IDH2/R140Q mutation is frequently detected in acute myeloid leukemia (AML). It contributes to leukemia via accumulation of oncometabolite D-2-HG. Therefore, mutant IDH2 is a promising target for AML. Discovery of IDH2 mutant inhibitors is in urgent need for AML therapy. Structure-based in silico screening and enzymatic assays were used to identify IDH2/R140Q inhibitors. Molecular docking, mutant structure building and molecular dynamics simulations were applied to investigate the inhibitory mechanism and selectivity of CP-17 on IDH2/R140Q. TF-1 cells overexpressed IDH2/R140Q mutant were used to study the effects of CP-17 on cellular proliferation and differentiation, the wild-type TF-1 cells were used as control. The intracellular D-2-HG production was measured by LC-MS. The histone methylation was evaluated with specific antibodies by western blot. CP-17, a heterocyclic urea amide compound, was identified as a potent inhibitor of IDH2/R140Q mutant by in silico screening and enzymatic assay. It exhibits excellent inhibitory activity with IC50 of 40.75 nM against IDH2/R140Q. More importantly, it shows poor activity against the wild-type IDH1/2, resulting in a high selectivity of over 55 folds, a dramatic improvement over previously developed inhibitors such as AGI-6780 and Enasidenib. Molecular simulations suggested that CP-17 binds to IDH2/R140Q at the allosteric site within the dimer interface through extensive polar and hydrophobic interactions, locking the enzyme active sites in open conformations with abolished activity to produce D-2-HG. Cellular assay results demonstrated that CP-17 inhibits intracellular D-2-HG production and suppresses the proliferation of TF-1 erythroleukemia cells carrying IDH2/R140Q mutant. Further, CP-17 also restores the EPO-induced differentiation that is blocked by the mutation and decreases hypermethylation of histone in the TF-1(IDH2/R140Q) cells. These results indicate that CP-17 can serve as a lead compound for the development of inhibitory drugs against AML with IDH2/R140Q mutant.

中文翻译：

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鉴定诱导白血病细胞分化的 IDH2/R140Q 酶的选择性抑制剂。

IDH2/R140Q 突变经常在急性髓性白血病 (AML) 中检测到。它通过致癌代谢物 D-2-HG 的积累导致白血病。因此，突变 IDH2 是 AML 的一个有希望的靶点。IDH2突变抑制剂的发现是AML治疗的迫切需要。基于结构的计算机筛选和酶分析用于鉴定 IDH2/R140Q 抑制剂。应用分子对接、突变结构构建和分子动力学模拟研究CP-17对IDH2/R140Q的抑制机制和选择性。用过表达IDH2/R140Q突变体的TF-1细胞研究CP-17对细胞增殖和分化的影响，野生型TF-1细胞作为对照。通过LC-MS测量细胞内D-2-HG的产生。通过蛋白质印迹用特异性抗体评估组蛋白甲基化。CP-17 是一种杂环脲酰胺化合物，通过计算机筛选和酶促测定被鉴定为 IDH2/R140Q 突变体的有效抑制剂。它对 IDH2/R140Q 表现出优异的抑制活性，IC50 为 40.75 nM。更重要的是，它显示出对野生型 IDH1/2 的低活性，导致超过 55 倍的高选择性，与先前开发的抑制剂如 AGI-6780 和 Enasidenib 相比有了显着改善。分子模拟表明，CP-17 通过广泛的极性和疏水相互作用在二聚体界面内的变构位点与 IDH2/R140Q 结合，将酶活性位点锁定在开放构象中，从而丧失产生 D-2-HG 的活性。细胞测定结果表明，CP-17 抑制细胞内 D-2-HG 的产生并抑制携带 IDH2/R140Q 突变体的 TF-1 红白血病细胞的增殖。此外，CP-17 还能恢复被突变阻断的 EPO 诱导的分化，并降低 TF-1 (IDH2/R140Q) 细胞中组蛋白的高甲基化。这些结果表明，CP-17 可作为开发针对具有 IDH2/R140Q 突变体的 AML 的抑制药物的先导化合物。